Dissecting the mechanism of epigenetic spreading by targeted degradation of architectural proteins In female mammals, one X chromosome is silenced in order to balance dosage of X-linked genes with males, a process known as X chromosome inactivation (XCI). XCI is mediated by a long noncoding RNA Xist, which spreads across the inactive X, triggering recruitment of repressive factors such as Polycomb Repressive Complexes 1 and 2 (PRC1/2), heterochromatin formation and gene silencing. In this way, XCI serves as a model for the spreading of heterochromatin marks, a process which is essential for gene silencing across the mammalian genome, but which is not well understood. XCI also dramatically alters the three-dimensional (3D) chromatin structure of the X chromosome. In particular, chromosome conformation studies revealed that the X chromosome, along with the rest of the mammalian genome is organized by architectural proteins CTCF and cohesin into Topologically Associating Domains (TADs), megabase-sized regions within which chromatin interactions form more frequently than across domain borders. Mammalian chromosomes are also organized independently of CTCF and cohesin into longer range ?compartmental? interactions between active and inactive regions (A/B compartments). While TADs and compartments generally remain stable during differentiation, both structures are dramatically altered during XCI. Specifically, TADs and compartments are greatly attenuated during XCI, with the inactive X instead being structured into megadomains. As such dramatic restructuring of chromosome conformation is not seen during any other cellular process, a pivotal question is whether these changes in X chromosome conformation are required for XCI or are merely a byproduct of silencing the entire chromosome. To address this question, I generated female mouse embryonic stem cells (mESCs) in which architectural proteins CTCF, RAD21, and WAPL can be rapidly degraded using the dTAG degron system during XCI. Using my CTCF and RAD21 degron cell lines, I first propose to weaken TADs and strengthen compartments in early XCI and study the impact of these changes on Xist spreading and subsequent steps of XCI. In contrast, I propose to use my WAPL degron cell lines to strengthen TADs and weaken compartments in early XCI and study the impact of these opposing changes on Xist spreading. By elucidating which, if any, 3D chromatin structures are needed for Xist spreading, I expect to shed light not only on XCI, but also on the mechanism of heterochromatin spreading outside of XCI?and the role of 3D genome organization in gene regulation in general as well.